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At least 19 recordsLinked to original sources
PubMed1999

Functional genomics and enzyme evolution. Homologous and analogous enzymes encoded in microbial genomes.

Computational analysis of complete genomes, followed by experimental testing of emerging hypotheses--the area of research often referred to as 'functional genomics'--aims at deciphering the wealth of information contained in genome sequences and at using it to improve our understanding of the mechanisms of cell function. This review centers on the recent progress in the genome analysis with special emphasis on the new insights in enzyme evolution. Standard methods of predicting functions for new proteins are listed and the common errors in their application are discussed. A new method of improving the functional predictions is introduced, based on a phylogenetic approach to functional prediction, as implemented in the recently constructed Clusters of Orthologous Groups (COG) database (available at http:@www.ncbi.nlm.nih.gov/COG). This approach provides a convenient way to characterize the protein families (and metabolic pathways) that are present or absent in any given organism. Comparative analysis of microbial genomes based on this approach shows that metabolic diversity generally correlates with the genome size-parasitic bacteria code for fewer enzymes and lesser number of metabolic pathways than their free-living relatives. Comparison of different genomes reveals another evolutionary trend, the non-orthologous gene displacement of some enzymes by unrelated proteins with the same cellular function. An examination of the phylogenetic distribution of such cases provides new clues to the problems of biochemical evolution, including evolution of glycolysis and the TCA cycle.

Databases, Factual↗
PubMed2004

Evolution of polyhydroxyalkanoate (PHA) production system by "enzyme evolution": successful case studies of directed evolution.

Biotechnological studies towards the biosynthesis of polyhydroxyalkanoates (PHAs) biopolyesters have extensively progressed through the development of various metabolic engineering strategies. Historically, efficient PHA production has been achieved using the fermentation technology of naturally occurring PHA-producing bacteria based on external substrate manipulation (1st generation), and subsequent reinforcement with recombinant gene technology (2nd generation). More recently, "enzyme evolution" is becoming the 3rd generation approach for PHA production. A break-through in the chemical synthesis of macromolecules with desirable properties was achieved by the development of prominent chemical catalysts via "catalyst evolution", as represented by a series of Ziegler-Natta catalysts. Thus, one can easily accept the concept that the molecular evolution of the biocatalysts (enzymes) relevant to PHA synthesis will provide us with a chance to create novel PHA materials with high performance. The first trial of an in vitro enzyme evolution in PHA biosynthesis was reported by our group in 2001. The following literature data, as well as our own experimental results devoted to this new approach, have been accumulated over a short time. This review article focuses specifically on the concept and current case studies of the application of "enzyme evolution" to PHA biosynthesis.

Acyltransferases↗
PubMed1997

Strategies for the in vitro evolution of protein function: enzyme evolution by random recombination of improved sequences.

Sets of genes improved by directed evolution can be recombined in vitro to produce further improvements in protein function. Recombination is particularly useful when improved sequences are available; costs of generating such sequences, however, must be weighed against the costs of further evolution by sequential random mutagenesis. Four genes encoding para-nitrobenzyl (pNB) esterase variants exhibiting enhanced activity were recombined in two cycles of high-fidelity DNA shuffling and screening. Genes encoding enzymes exhibiting further improvements in activity were analyzed in order to elucidate evolutionary processes at the DNA level and begin to provide an experimental basis for choosing in vitro evolution strategies and setting key parameters for recombination. DNA sequencing of improved variants from the two rounds of DNA shuffling confirmed important features of the recombination process: rapid fixation and accumulation of beneficial mutations from multiple parent sequences as well as removal of silent and deleterious mutations. The five to sixfold further enhancement of total activity towards the para-nitrophenyl (pNP) ester of loracarbef was obtained through recombination of mutations from several parent sequences as well as new point mutations. Computer simulations of recombination and screening illustrate the trade-offs between recombining fewer parent sequences (in order to reduce screening requirements) and lowering the potential for further evolution. Search strategies which may substantially reduce screening requirements in certain situations are described.

Carboxylic Ester Hydrolases↗
PubMed2006

Recent advances in biocatalysis by directed enzyme evolution.

Naturally occurring enzymes are remarkable biocatalysts with numerous potential applications in industry and medicine. However, many of their catalyst properties often need to be further tailored to meet the specific requirements of a given application. Within this context, directed evolution has emerged over the past decade as a powerful tool for engineering enzymes with new or improved functions. This review summarizes recent advances in applying directed evolution approaches to alter various enzyme properties such as activity, selectivity (enantio- and regio-), substrate specificity, stability, and solubility. Special attention will be paid to the creation of novel enzyme activities and products by directed evolution.

Bacteria↗
PubMed2001

In vitro enzyme evolution: the screening challenge of isolating the one in a million.

The generation of large mutant libraries for in vitro enzyme evolution presents the challenge of effectively screening libraries of 10(4)-10(7) mutants on the basis of simultaneously assaying their biocatalytic activity. In this review, we highlight the main steps involved in this process, describe the alternative approaches to address this challenge, survey the state-of-the-art technology and assess achievements already made. It is anticipated that, as a result of the expected accomplishment of further improvements in high-throughput screening that will allow routine screening of whole libraries, the number of useful new and improved enzymes derived through in vitro enzyme evolution will expand rapidly in the near future.

Cells, Immobilized↗
PubMed2004

Network analysis of metabolic enzyme evolution in Escherichia coli.

BACKGROUND: The two most common models for the evolution of metabolism are the patchwork evolution model, where enzymes are thought to diverge from broad to narrow substrate specificity, and the retrograde evolution model, according to which enzymes evolve in response to substrate depletion. Analysis of the distribution of homologous enzyme pairs in the metabolic network can shed light on the respective importance of the two models. We here investigate the evolution of the metabolism in E. coli viewed as a single network using EcoCyc. RESULTS: Sequence comparison between all enzyme pairs was performed and the minimal path length (MPL) between all enzyme pairs was determined. We find a strong over-representation of homologous enzymes at MPL 1. We show that the functionally similar and functionally undetermined enzyme pairs are responsible for most of the over-representation of homologous enzyme pairs at MPL 1. CONCLUSIONS: The retrograde evolution model predicts that homologous enzymes pairs are at short metabolic distances from each other. In general agreement with previous studies we find that homologous enzymes occur close to each other in the network more often than expected by chance, which lends some support to the retrograde evolution model. However, we show that the homologous enzyme pairs which may have evolved through retrograde evolution, namely the pairs that are functionally dissimilar, show a weaker over-representation at MPL 1 than the functionally similar enzyme pairs. Our study indicates that, while the retrograde evolution model may have played a small part, the patchwork evolution model is the predominant process of metabolic enzyme evolution.

Algorithms↗
PubMed2001

Structure-based chimeric enzymes as an alternative to directed enzyme evolution: phytase as a test case.

Thermostability is a key feature for commercially attractive variants of the fungal enzyme phytase. In an initial set of experiments, we restored ionic interactions and hydrogen bonds on the surface of Aspergillus terreus phytase, which are present in the homologous but more thermostable enzyme from A. niger. Since these mutations turned out to be neutral, we replaced-in the same region and based on the crystal structure of A. niger phytase-entire secondary structure elements. The replacement of one alpha-helix on the surface of A. terreus phytase by the corresponding stretch of A. niger phytase resulted in an enzyme with improved thermostability and unaltered enzymatic activity. Surprisingly, the thermostability of this hybrid protein was very similar to that of A. niger phytase, although the fusion protein contained only a 31 amino acid stretch of the more stable parent enzyme. This report provides evidence that structure-based chimeric enzymes can be used to exploit the evolutionary information within a sequence alignment. We propose this method as an alternative to directed enzyme evolution if due to expression constraints the screening of large mutant populations is not feasible.

Amino Acid Sequence↗
PubMed1993

Critical amino acids responsible for converting specificities of proteins and for enhancing enzyme evolution are located around beta-turn potentials: data-based prediction.

Various reports have described that amino acid substitutions can alter substrate, positional, inhibitory, and target gene specificities of proteins. By using the method of Chou and Fasman, the present work predicts that critical amino acids for converting these substrate specificities of trypsin, L-lactate dehydrogenase, aspartate aminotransferase, beta-lactamase, and cytochrome P-450 are found to exist within regions predicted as beta-turns. The ratios of hydroxylation and oxygenation positions of substrates by cytochrome P-450 and lipoxygenase, respectively, are varied by changes of the protein structures, probably around turn conformations. Inhibitory specificities of bovine pancreatic trypsin inhibitor and alpha 1-antitrypsin and target gene specificity of glucocorticoid receptor are converted by changing turn structures. Occurrence of beta-turn probabilities can be predicted around the amino acid alteration positions of an evolutionally antecedent protein of a nylon degradation enzyme. These findings will have relevance to work on protein engineering and enzyme evolution.

Amino Acid Sequence↗
PubMed2004

Directed enzyme evolution and selections for catalysis based on product formation.

Enzyme engineering by molecular modelling and site-directed mutagenesis can be remarkably efficient. Directed enzyme evolution appears as a more general strategy for the isolation of catalysts as it can be applied to most chemical reactions in aqueous solutions. Selections, as opposed to screening, allow the simultaneous analysis of protein properties for sets of up to about 10(14) different proteins. These approaches for the parallel processing of molecular information 'Is the protein a catalyst?' are reviewed here in the case of selections based on the formation of a specific reaction product. Several questions are addressed about in vivo and in vitro selections for catalysis reported in the literature. Can the selection system be extended to other types of enzymes? Does the selection control regio- and stereo-selectivity? Does the selection allow the isolation of enzymes with an efficient turnover? How should substrates be substituted or mimicked for the design of efficient selections while minimising the number of chemical synthesis steps? Engineering sections provide also some clues to design selections or to circumvent selection biases. A special emphasis is put on the comparison of in vivo and in vitro selections for catalysis.

Biotechnology↗
PubMed2001

Catalytic and binding poly-reactivities shared by two unrelated proteins: The potential role of promiscuity in enzyme evolution.

It is generally accepted that enzymes evolved via gene duplication of existing proteins. But duplicated genes can serve as a starting point for the evolution of a new function only if the protein they encode happens to exhibit some activity towards this new function. Although the importance of such catalytic promiscuity in enzyme evolution has been proposed, little is actually known regarding how common promiscuous catalytic activities are in proteins or their origins, magnitudes, and potential contribution to the survival of an organism. Here we describe a pattern of promiscuous activities in two completely unrelated proteins-serum albumins and a catalytic antibody (aldolase antibody 38C2). Despite considerable structural dissimilarities-in the shape of the cavities and the position of catalytic lysine residues-both active sites are able to catalyze the Kemp elimination, a model reaction for proton transfer from carbon. We also show that these different active sites can bind promiscuously an array of hydrophobic negatively charged ligands. We suggest that the basic active-site features of an apolar pocket and a lysine residue can act as a primitive active site allowing these promiscuous activities to take place. We also describe, by modelling product formation at different substrate concentrations, how promiscuous activities of this kind- inefficient and rudimentary as they are-can provide a considerable selective advantage and a starting point for the evolution of new functions.

Binding Sites↗
PubMed1981

Variability of electrocardiographic and enzyme evolution of myocardial infarction in man.

We have studied the time course of development of ST segment elevation, R wave loss, and Q wave development in 41 patients using 35 lead praecordial mapping or 12 lead electrocardiograms in those with anterior and inferior infarcts, respectively. The first recording was at a mean time of six hours after the onset of pain; subsequent records were taken every eight hours for 24 hours, every 12 hours for the second day, and every day thereafter. Serial CK MB estimates were obtained at every four hours for the first 72 hours. There was good agreement in the time course between the electrocardiogram and enzyme evolution. Forty-one per cent of patients showed rapid infarction with R wave and Q wave evolution complete within 12 hours of pain and accompanied by a short duration of enzyme release (mean = 19.30 hours). Fifty-nine per cent of patients showed more prolonged infarction with longer R wave and Q wave evolution and enzyme release (mean = 30 hours). Four patients also showed delayed reinfarction. ST segment elevation was maximal at six hours in the whole group and was significantly lower thereafter. Patients with rapid infarction showed high initial ST segment elevation which decreased promptly compared with those with prolonged infarction, who showed moderate but more persistent ST segment elevation. This study shows the variability in the time course of the electrocardiogram and enzyme evolution after myocardial infarction in man.

Aged↗
PubMed1972

Enzyme evolution. I. The importance of untranslatable intermediates.

The proportion of two allelic mutant forms of a haploid species can be calculated from six quantities, the original proportion, the two effective growth rate constants, the two mutation rate constants for the transition from one to the other, and the elapsed time. With a program for carrying out this calculation in a mini-computer, we set about investigating the rates of exploration of multiple intragene changes in populations of enormous numbers over a large number of cell generations. It seemed evident from the outset that unless in the past very different proportions of the different types of mutational changes occurred, then neither the numbers nor the times could have been of sufficient magnitude to account for enzyme evolution by multiple simultaneous changes within a gene to yield a superior protein. Rather, we propose that the more rapid path of enzyme evolution consisted of the sequence: 1) gene duplication, 2) inactivation of a gene copy by a chain-terminating mutation or other mutational change which suppresses transcription and/or translation, 3) multiple base-pair changes and/or small deletions and additions within the non-functioning gene, and finally, 4) reversion of the mutation which was suppressing translation. It is shown that this path will lead to faster advance, if the selective advantages of different forms are dependent on the function of the gene products at certain times in history, while at other times, although this function may be necessary, it is not limiting for growth.

Alleles↗
PubMed1993

Comparison of the nucleotide sequences of the meta-cleavage pathway genes of TOL plasmid pWW0 from Pseudomonas putida with other meta-cleavage genes suggests that both single and multiple nucleotide substitutions contribute to enzyme evolution.

TOL plasmid pWW0 from Pseudomonas putida mt-2 encodes catabolic enzymes required for the oxidation of toluene and xylenes. The structural genes for these catabolic enzymes are clustered into two operons, the xylCMABN operon, which encodes a set of enzymes required for the transformation of toluene/xylenes to benzoate/toluates, and the xylXYZLTEGFJQKIH operon, which encodes a set of enzymes required for the transformation of benzoate/toluates to Krebs cycle intermediates. The latter operon can be divided physically and functionally into two parts, the xylXYZL cluster, which is involved in the transformation of benzoate/toluates to (methyl)catechols, and the xylTEGFJQKIH cluster, which is involved in the transformation of (methyl)catechols to Krebs cycle intermediates. Genes isofunctional to xylXYZL are present in Acinetobacter calcoaceticus, and constitute a benzoate-degradative pathway, while xylTEGFJQKIH homologous encoding enzymes of a methylphenol-degradative pathway and a naphthalene-degradative pathway are present on plasmid pVI150 from P. putida CF600, and on plasmid NAH7 from P. putida PpG7, respectively. Comparison of the nucleotide sequences of the xylXYZLTEGFJQKIH genes with other isofunctional genes suggested that the xylTEGFJQKIH genes on the TOL plasmid diverged from these homologues 20 to 50 million years ago, while the xylXYZL genes diverged from the A. calcoaceticus homologues 100 to 200 million years ago. In codons where amino acids are not conserved, the substitutions rate in the third base was higher than that in synonymous codons. This result was interpreted as indicating that both single and multiple nucleotide substitutions contributed to the amino acid-substituting mutations, and hence to enzyme evolution. This observation seems to be general because mammalian globin genes exhibit the same tendency.

Amino Acid Sequence↗
PubMed1997

Proteins, RNAs and chaperones in enzyme evolution: a folding perspective.

The present roles of RNA molecules as templates and of proteins as cellular catalysts may not have always been so clearly defined during evolution. Recent work on ribozymes shows that the catalytic activity of early RNAs may have been more general than previously thought. How did evolution select proteins, not RNA, to be the major biological catalysts? Why were chaperones necessary for the evolution of modern protein enzymes?

Biological Evolution↗
PubMed2001

Evolving hardware as model of enzyme evolution.

Organism growth and survival is based on thousands of enzymes organized in networks. The motivation to understand how a large number of enzymes evolved so fast inside cells may be relevant to explaining the origin and maintenance of life on Earth. This paper presents electronic circuits called 'electronic enzymes' that model the catalytic function performed by biological enzymes. Electronic enzymes are the hardware realization of enzymes defined as molecular automata with a finite number of internal conformational states and a set of Boolean operators modelling the active groups of the active site. One of the main features of electronic enzymes is the possibility of evolution finding the proper active site by means of a genetic algorithm yielding a metabolic ring or k-cycle that bears a resemblance to Krebs (k=7) or Calvin (k=4) cycles present in organisms. The simulations are consistent with those results obtained in vitro evolving enzymes based on polymerase chain reaction (PCR) as well as with the general view that suggests the main role of recombination during enzyme evolution. The proposed methodology shows how molecular automata with evolvable features that model enzymes or other processing molecules provide an experimental framework for simulation of the principles governing metabolic pathways evolution and self-organization.

Algorithms↗